Metazoans differentially express multiple Hox transcription factors to specify diverse cell fates along the developing anterior-posterior axis. Two challenges arise when trying to understand how the ...Hox transcription factors regulate the required target genes for morphogenesis: First, how does each Hox factor differ from one another to accurately activate and repress target genes required for the formation of distinct segment and regional identities? Second, how can a Hox factor that is broadly expressed in many tissues within a segment impact the development of specific organs by regulating target genes in a cell type-specific manner? In this review, we highlight how recent genomic, interactome, and
cis
-regulatory studies are providing new insights into answering these two questions. Collectively, these studies suggest that Hox factors may differentially modify the chromatin of gene targets as well as utilize numerous interactions with additional co-activators, co-repressors, and sequence-specific transcription factors to achieve accurate segment and cell type-specific transcriptional outcomes.
Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in ...Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.
cis-regulatory modules (CRMs) generate precise expression patterns by integrating numerous transcription factors (TFs). Surprisingly, CRMs that control essential gene patterns can differ greatly in ...conservation, suggesting distinct constraints on TF binding sites. Here, we show that a highly conserved Distal-less regulatory element (DCRE) that controls gene expression in leg precursor cells recruits multiple Hox, Extradenticle (Exd) and Homothorax (Hth) complexes to mediate dual outputs: thoracic activation and abdominal repression. Using reporter assays, we found that abdominal repression is particularly robust, as neither individual binding site mutations nor a DNA binding deficient Hth protein abolished cooperative DNA binding and in vivo repression. Moreover, a re-engineered DCRE containing a distinct configuration of Hox, Exd, and Hth sites also mediated abdominal Hox repression. However, the re-engineered DCRE failed to perform additional segment-specific functions such as thoracic activation. These findings are consistent with two emerging concepts in gene regulation: First, the abdominal Hox/Exd/Hth factors utilize protein-protein and protein-DNA interactions to form repression complexes on flexible combinations of sites, consistent with the TF collective model of CRM organization. Second, the conserved DCRE mediates multiple cell-type specific outputs, consistent with recent findings that pleiotropic CRMs are associated with conserved TF binding and added evolutionary constraints.
Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss ...of enteroendocrine cells, whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3−/− pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues, whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability, DNA binding, and gene transcription. Moreover, NEUROG3 was highly unstable in the intestinal epithelium, explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships.
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•Human PSC-derived pancreas and intestinal endocrine cells to model NEUROG3 mutations•NEUROG3 mutations affect protein stability, dimerization, and DNA binding•NEUROG3 protein is less stable in intestinal epithelium than in the pancreas•Reduced protein stability in intestine enhances sensitivity to NEUROG3 mutations
Zhang et al. use a human pluripotent stem cell-based system to study the impact of patient-derived mutations in NEUROG3 on the development of pancreatic and intestinal endocrine cells. This system recapitulates patient phenotypes, identifies hypomorphic alleles, and uncovers the mutations’ effects on dimerization, DNA binding, transcription, and protein stability.
Superior manual dexterity in higher primates emerged together with the appearance of cortico-motoneuronal (CM) connections during the evolution of the mammalian corticospinal (CS) system. Previously ...thought to be specific to higher primates, we identified transient CM connections in early postnatal mice, which are eventually eliminated by Sema6D-PlexA1 signaling. PlexA1 mutant mice maintain CM connections into adulthood and exhibit superior manual dexterity as compared with that of controls. Last, differing PlexA1 expression in layer 5 of the motor cortex, which is strong in wild-type mice but weak in humans, may be explained by FEZF2-mediated cis-regulatory elements that are found only in higher primates. Thus, species-dependent regulation of PlexA1 expression may have been crucial in the evolution of mammalian CS systems that improved fine motor control in higher primates.
Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and ...oncoprotein YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase LATS/Warts (Wts) and growth regulator Melted generates mutually exclusive photoreceptor subtypes. Here, we show that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network-including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module-that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution.
Hox transcription factors specify distinct cell types along the anterior-posterior axis of metazoans by regulating target genes that modulate signaling pathways. A well-established example is the ...induction of Epidermal Growth Factor (EGF) signaling by an Abdominal-A (Abd-A) Hox complex during the specification of Drosophila hepatocyte-like cells (oenocytes). Previous studies revealed that Abd-A is non-cell autonomously required to promote oenocyte fate by directly activating a gene (rhomboid) that triggers EGF secretion from sensory organ precursor (SOP) cells. Neighboring cells that receive the EGF signal initiate a largely unknown pathway to promote oenocyte fate. Here, we show that Abd-A also plays a cell autonomous role in inducing oenocyte fate by activating the expression of the Pointed-P1 (PntP1) ETS transcription factor downstream of EGF signaling. Genetic studies demonstrate that both PntP1 and PntP2 are required for oenocyte specification. Moreover, we found that PntP1 contains a conserved enhancer (PntP1OE) that is activated in oenocyte precursor cells by EGF signaling via direct regulation by the Pnt transcription factors as well as a transcription factor complex consisting of Abd-A, Extradenticle, and Homothorax. Our findings demonstrate that the same Abd-A Hox complex required for sending the EGF signal from SOP cells, enhances the competency of receiving cells to select oenocyte cell fate by up-regulating PntP1. Since PntP1 is a downstream effector of EGF signaling, these findings provide insight into how a Hox factor can both trigger and potentiate the EGF signal to promote an essential cell fate along the body plan.
Metazoans encode clusters of paralogous Hox genes that are critical for proper development of the body plan. However, there are a number of unresolved issues regarding how paralogous Hox factors ...achieve specificity to control distinct cell fates. First, how do Hox paralogs, which have very similar DNA binding preferences
, drive different transcriptional programs
? Second, the number of potential Hox binding sites within the genome is vast compared to the number of sites bound. Hence, what determines where in the genome Hox factors bind? Third, what determines whether a Hox factor will activate or repress a specific target gene? Here, we review the current evidence that is beginning to shed light onto these questions. In particular, we highlight how cooperative interactions with other transcription factors (especially PBC and HMP proteins) and the sequences of
-regulatory modules provide a basis for the mechanisms of Hox specificity. We conclude by integrating a number of the concepts described throughout the review in a case study of a highly interrogated Drosophila
-regulatory module named "The Distal-less Conserved Regulatory Element" (DCRE).
Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF ...binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs.
Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the ...Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.
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